1. Field of the Invention
The present invention relates to a tumor specific, mitogenic growth factor from platelets, its purification, and its uses.
2. Description of the Prior Art
Cultivation of most mammalian cells as currently practiced in vitro requires a complex mixture of nutrients, including macromolecules of unknown structure isolated from biological sources. Presently, cell lines are grown in media containing serum, and particularly those which contain fetal calf serum. However, the relative scarcity and expense of fetal calf serum has led to the investigation of other media in which serum is not required. In addition, since the exact nature of all components in serum is unknown, it has been impossible to specify exactly which components are required for growth of cells. Accordingly, attempts to study cell growth have been hampered by the lack of growth media having a defined composition.
Various studies have shown that, in addition to simple nutrients necessary for the growth of any simple organism, differentiated cells such as tissue cells require more complex substances for growth or cell division, presumably synthesized by other parts of the organism in which the tissue is found. These substances, whose structures are generally unknown, are referred to as growth factors.
Previous work in this area has shown that growth factors present in blood serum promote the multiplication and survival of certain cultivated cells, such as 3T3 mouse fibroblasts. Various growth factors have been isolated which are indicated to be of use in promoting cell division of non-transformed, i.e., normal, cells: for example, a growth-promoting polypeptide isolated from blood serum and disclosed in U.S. Pat. No. 4,054,557. However, also desirable are growth factors, for transformed cells, i.e., those cells that have been converted by a viral or chemical agent into a tumor-like cell line. Such cells no longer grow in orderly fashion in cell cultures and result in tumors when injected into animals. Isolation of tumor-specific growth factors would aid in the study of in vivo and in vitro tumor growth and be of benefit in the clinical treatment of tumors.
One evident source of cell growth factors is blood serum, since blood serum itself will support cell growth in vitro. Blood serum, the liquid fraction of blood that remains after clotting, has been used as a major component of tissue culture media. Blood plasma, the liquid fraction of blood that has not been allowed to clot, has been shown to be less effective than serum in promoting the growth of various tissue culture lines, such as chicken or mouse fibroblasts. Plasma does, however, allow for the survival of cells without an increase in their cell number.
Accordingly, it has been postulated that a serum mitogenic (cell division) factor, or factors, is released from precursors in plasma or from platelets (thrombocytes) when blood is clotted in the preparation of serum. This hypothesis has been tested in previous studies by both of the present inventors, some of which are reported in Kohler and Lipton, Experimental Cell Research 87 (1974) 297-301 and Kepner, Creasy, and Lipton, "Platelets as a Source of Cell-Proliferating Activity" in Platelets, de Gaetano and Garattini, eds., Raven Press, N.Y. (1978), which are hereby incorporated by reference. Serum and plasma were separately fractionated on Sephadex G-100 columns at pH 7.4 and pH 2. Sephadex is a bead-formed gel prepared by cross-linking dextran with epichlorohydrin. Sephadex G-100 comprises beads of 40-120 .mu.m diameter (dry) and having a pore size selective for peptides and globular proteins in the molecular weight range of 4,000 to 150,000 daltons and dextrans in the range 1,000 to 100,00 daltons.
The growth promoting activity for 3T3 mouse fibroblasts from rat serum and plasma appeared to be eluted at the same time from each column. There was, however, a greater stimulation of cell growth with serum eluent than with plasma eluent. The growth promoting factor(s) found in plasma appeared to be similar in molecular weight to that found in serum. Therefore, it did not appear that the serum growth factor was the product of degradation of a precursor of higher molecular weight present in plasma. It seemed likely that the mitogenic activity for 3T3 cells found in serum was instead released from platelets during the clotting process. This work was reported in Kohler and Lipton, Experimental Cell Research 87 (1974) 297-301.
Platelets were next shown to be a direct source of mitogenic activity. Human platelet extracts were made by freezing and thawing the cells six times in glass tubes to disrupt the platelets. The platelet extract prepared in this fashion stimulated the growth of 3T3 cells approximately 5-fold more than an equivalent amount of serum. This same platelet extract was also used in a culture medium for SV3T3 cells, mouse fibroblasts that had been transformed by Simian virus into a tumor-like cell line. There appeared to be little difference in stimulation for this cell line between the human platelet extract and human serum, an indication that the extract at this stage was not specific for tumors. This initial platelet extract was designated PDGF.sub.1, (platelet derived growth factor for non-transformed cells), and is described in Kohler and Lipton, Experimental Cell Research 87 (1974) 297-301.
In an attempt to isolate growth factors specific for tumors, other extraction techniques were tried with platelets. Mitogenic activity for SV3T3 cells could be obtained by washing outdated human platelets with physiological saline or by incubating thus washed platelets with 10.sup.-8 M ADP (or thrombin or N-ethylmaleimide) in the absence of plasma and calcium. Unlike the original frozen and thawed extracts, both platelet saline wash and ADP treated platelet supernatants (ADP released factor) were more active than serum in promoting the growth and survival of SV3T3 cells.
Gel filtration of either the saline wash material or the ADP release factor on Sephadex G-100 at pH 7.4 yielded a stimulating fraction for SV3T3 cells having a molecular weight of approximately 72,000 daltons and an isoelectric point of approximately 10-10.5. Activity was lost when the platelet extracts were treated with periodate, indicating the presence of a glycoprotein. This factor was designated PDGF.sub.T (platelet dependent growth factor for transformed cells) and is described in an article by Kepner, Creary, and Lipton in Platelets, de Gaetano and Gasattini, eds. Raven Press, N.Y. (1978) p. 205-212. However, this platelet extract represents only a 1- or 2-fold purification over serum, and, accordingly, does not represent a desirable agent for use in a purified, tissue-cell growth medium. In the process of working toward isolation of a pure growth factor, the present invention was made.